The goal is to develop microchip-based IEF coupled with in situ introduction of matrix solution, providing the protein/matrix mixtures which are amenable to direct MALDI-MS analysis. IEF separates proteins on the basis of their differences in isoelectric point. Once the focusing is complete in the lower microchannel of a 3-dimensional (3-D) MEMS, a matrix solution is introduced into the upper flow channel. The upper flow channel is vertically isolated from the lower microchannel by a movable partition plate. This plate is actuated through an off-chip piezoelectric element, forcing the plate to slide laterally for the creation of a narrow opening between the channels. This displacement permits direct mixing of matrix solution with the underlying focused protein bands, while avoiding disturbance in the longitudinal resolution of proteins. The proteins within the matrix crystals can be characterized using MALDI-MS with extremely high sensitivity, wide mass range, and high tolerance to impurities. In analogy to a 2-D analysis system, fundamental studies on the fabrication of 3-D MEMS and the performance of isoelectric focusing in the microchannel establish the basis for the first separation mechanism. Basic understanding of in situ introduction of matrix solution and the evaluation of MALDI-MS performance in the presence of various separation additives serve as the second separation dimension. The on-chip introduction of matrix solution in an automated fashion obviates the need to mobilize and electroblot proteins as in the current capillary and gel based electrophoretic separations. Building on experience gained from this proposed research, the simultaneous separation of analyte proteins in the array of miniaturized IEF together with MALDI-MS analysis may allow high throughput monitoring of protein expression, including posttranslational modifications and their changes under different physiological and pathophysiological conditions. The resulting technology will also enable the investigators to approach the high throughput analysis of the corresponding proteomes from the Human Genome Project.